Structural basis for transmembrane Mg2+ transport Abstract Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers and promoter of genomic stability, among other functions. In this proposal we will focus on the CorA Mg2+ transporter/channel, which functions as the primary Mg2+ uptake system for Eubacteria and Archaea. The structure of the CorA ortholog from Thermotoga maritima has been recently determined at medium resolution, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ transport and regulation in prokaryotic mechanosensitive channels. Although the recent determination of the CorA crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel/transport gating. In this respect, we plan to experimentally address several fundamental questions: Is CorA a coupled transporter of an ion channel? What regions of CorA form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the structures of the key functional states? The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally-similar orthologs will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis o the mechanisms of ion translocation and gating, and signal transduction. PUBLIC HEALTH RELEVANCE: Understanding of CorA structure and function relates directly to health and disease, not only as key element in the most basic aspect of cellular function but due to its relationship to the mechanism of mitochondrial Mg++ homeostasis in eukaryotic cells. This is relevant because of the known role of mitochondria in apoptosis. CorA is also a virulence factor in prokaryotes and thus an important potential antibiotic target.